molecules

Article
Antiviral Activity of Metabolites from Peruvian Plants against
SARS-CoV-2: An In Silico Approach
Luis Daniel Goyzueta-Mamani 1, * , Haruna Luz Barazorda-Ccahuana 1, * , Karel Mena-Ulecia 2,3
and Miguel Angel Chávez-Fumagalli 1

1 Vicerrectorado de Investigación, Universidad Católica de Santa María, Urb. San José s/n—Umacollo,
Arequipa 04000, Peru; mchavezf@ucsm.edu.pe
2 Departamento de Ciencias Biológicas y Químicas, Facultad de Recursos Naturales,
Universidad Católica de Temuco, Ave. Rudecindo Ortega 02950, Temuco 4780000, Chile; kmena@uct.cl
3 Núcleo de Investigación en Bioproductos y Materiales Avanzados (BIOMA), Facultad de Recursos Naturales,
Universidad Católica de Temuco, Ave. Rudecindo Ortega 02950, Temuco 4780000, Chile
* Correspondence: lgoyzueta@ucsm.edu.pe (L.D.G.-M.); hbarazorda@ucsm.edu.pe (H.L.B.-C.)






Citation: Goyzueta-Mamani, L.D.;
Barazorda-Ccahuana, H.L.;
Mena-Ulecia, K.; Chávez-Fumagalli,
M.A. Antiviral Activity of
Metabolites from Peruvian Plants
against SARS-CoV-2: An In Silico
Approach. Molecules 2021, 26, 3882.
https://doi.org/10.3390/
molecules26133882
Abstract: (1) Background: The COVID-19 pandemic lacks treatments; for this reason, the search
for potential compounds against therapeutic targets is still necessary. Bioinformatics tools have
allowed the rapid in silico screening of possible new metabolite candidates from natural resources
or repurposing known ones. Thus, in this work, we aimed to select phytochemical candidates from
Peruvian plants with antiviral potential against three therapeutical targets of SARS-CoV-2. (2) Meth-
ods: We applied in silico technics, such as virtual screening, molecular docking, molecular dynamics
simulation, and MM/GBSA estimation. (3) Results: Rutin, a compound present in Peruvian native
plants, showed affinity against three targets of SARS-CoV-2. The molecular dynamics simulation
demonstrated the high stability of receptor–ligand systems during the time of the simulation. Our re-
sults showed that the Mpro-Rutin system exhibited higher binding free energy than PLpro-Rutin
and N-Rutin systems through MM/GBSA analysis. (4) Conclusions: Our study provides insight on
natural metabolites from Peruvian plants with therapeutical potential. We found Rutin as a potential
candidate with multiple pharmacological properties against SARS-CoV-2.
Keywords: in silico; natural products; Rutin; Peru; SARS-CoV-2; Bioinformatics; drug discovery

Academic Editors: Maria Atanassova

and Mark Brönstrup
1. Introduction
Received: 15 May 2021
In the past, different pandemics have affected the health and economy of nations,
Accepted: 21 June 2021
such as Cholera, Spanish flu (caused by the H1N1 virus), Severe acute respiratory syndrome
Published: 25 June 2021
(SARS, caused by SARS-CoV virus), the Middle East respiratory syndrome (MERS, caused
by MERS-CoV virus) [1], and, recently, the COVID-19 pandemic, caused by the novel
Publisher’s Note: MDPI stays neutral
coronavirus SARS-CoV-2. This new pandemic emerged in China in late 2019 and spread
with regard to jurisdictional claims in
rapidly, infecting 172 million people and causing 3.7 million deaths worldwide up to June
published maps and institutional affil-
iations.
of 2021 [2,3].
The symptoms of SARS-CoV-2 infection vary from typical flu-like symptoms to severe
pneumonia. Some common symptoms are fever, cough, shortness of breath or difficulty
breathing, muscular aches, and possible loss of taste or smell [4]. The detection of infected
individuals and the accurate diagnosis must be performed to contain the rapid disease
Copyright: © 2021 by the authors.
expansion. There are several options applied routinely, such as immunological based
Licensee MDPI, Basel, Switzerland.
analysis (i.e., Enzyme-linked immunosorbent assay (ELISA), lateral flow), imaging-based
This article is an open access article
(i.e., computed tomography (CT)), and molecular biology-based (i.e., Reverse transcription-
distributed under the terms and
conditions of the Creative Commons
polymerase chain reaction (RT-PCR), loop-mediated isothermal amplification (LAMP),
Attribution (CC BY) license (https://
clustered regularly interspaced short palindromic repeats (CRISPR), and Specific high-
creativecommons.org/licenses/by/ sensitivity enzymatic reporter unlocking (SHERLOCK)) [5]. Although RT-PCR is the most
4.0/).

Molecules 2021, 26, 3882. https://doi.org/10.3390/molecules26133882 https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 3882 2 of 17

accurate test among the others, it still has some challenges and issues that difficult its
validation and specificity, such as false negatives and viral loads [6].
To date, the Food and Drug Administration (FDA) approved three vaccines for their
emergency use (Pfizer-BioNTech, Moderna, and Janssen), and other 14 vaccines were
approved for their use in at least one country [7]. According to the Our World in Data
website [8], 272 million people were fully vaccinated (3.5% of the world’s population).
Unfortunately, the efficacy study of the vaccines against new variants, people from various
age groups, and comorbidities is still ongoing and needs validation. In addition, the FDA
had only emergency-approved antiviral drugs such as remdesivir, baricitinib, and the
monoclonal antibody bamlanivimab [9]. However, due to their availability and cost,
there are no accessible options for mass treatment [10]. In this sense, new drugs and
accessible treatments worldwide against this novel virus remains a challenge [11].
Drug repositioning is the process of discovering, validating, and marketing from
previously approved drugs for other indications, which can be used as a fast-track approach
guided by established target product profiles. As the new indication is built on already
available pharmacokinetics and safety data, the drug development time and cost can
be shortened and manufacturing issues can be better defined [12]. The study and trial
of repositioning already-approved, such as the protease inhibitors lopinavir, indinavir,
and ritonavir [13,14]. In the same way, the in vitro test results of these inhibitors against
SARS and MERS viruses were promising but remain unclear [15]. According to the National
Institutes of Health (NIH), there are 71 active studies in phases III and IV of clinical trials
about the treatment and prevention of COVID-19 [16].
The SARS-CoV-2 virus belongs to the betacoronavirus genus, which is characterized by
different structural proteins, such as the matrix protein (M), spike surface glycoprotein (S),
hemagglutinin-esterase dimer protein, a small envelope protein (E), and N-terminal RNA
binding domain of nucleocapsid protein (N); these proteins are attractive as therapeutic
molecular targets for drug and vaccine development [17]. Furthermore, the main protease
(Mpro), a protein with no human homolog, mediates the functional maturation of polypep-
tides related to the replication–transcription machinery assembly [18]. The Papain-like
protease (PLpro) is an interferon (IFN) antagonist that constitutes a domain of the replicase
polyprotein and may be active at an early stage of the replication cycle to antagonize an
upstream step of IFN induction [19]. The nucleocapsid protein (N), which encapsulates
the viral genome protecting it from the host cell environment and enhances the efficiency
of sub-genomic viral RNA transcription, is vital for viral replication; these are promising
molecular targets that are also being extensively studied and used for drug and vaccine
development [20].
Natural products have traditionally played a key role in drug discovery and were the
basis of most early medicines [21]. According to Newman and Cragg (2020) [22], the num-
ber of natural product-derived drugs present in the total amount of drug launchings in the
market from 1981 to 2019 represented about 42%, primarily obtained from plant sources.
Therefore, bioinformatics and computational approaches became crucial for rapid in silico
screening of potential metabolite databases from natural sources that can be repurposed
against SARS-CoV-2 for faster, safer, and cheaper drug development reported antiviral ac-
tivities [23]. These simulations can be carried out against proteins, enzymes, and receptors
involved in the life cycle of the SARS-CoV-2 virus.
In this context, Peru possesses 28 of the 32 current climates in the world and 84 of the
103 life zones known on earth [24]. It is considered one of the 12 megadiverse countries,
with a varied flora calculated in approximately 25,000 species [25]. Thus, ~10% of the
world’s flora grows in Peru, and 30% of these plants are endemic. The Peruvian population
uses approximately 5000 Peruvian plants for 49 purposes or applications (1400 species
are described as medicinal) [26,27]. Examples of these natural sources are the following
most exported native vegetal species: Uncaria tomentosa, commonly known as Cat’s claw,
used due to its anti-inflammatory effect, possibly due to the presence of proanthocyani-
dins [28]; the sap from Croton lechleri, commonly known as Sangre de Grado, is used due to
Molecules 2021, 26, 3882 3 of 17

their anti-inflammatory, antiseptic, and hemostatic properties attributed to the presence of

oligomeric proanthocyanidins, taspine, and flavonoids [29,30]; and Plukenetia volubilis L.
known as Sacha inchi, used as a nutraceutical food due to its high-quality content in tria-
cylglycerols, polyphenols and tocopherols [31,32]. However, most studies reported crude
medicinal activities, while potentially active compounds have been isolated only from a
few of the plants tested [33]. In this sense, the amount and nature of experimental evidence
published on active compounds isolated from Peruvian plants are still limited [34].
Considering all the properties and understanding the importance and immense value
of the in silico and computer-aided drug screening, this work aimed to select potential
phytochemical candidates from Peruvian plants with antiviral activity against three thera-
peutical SARS-CoV-2 targets. We have used bibliographic analysis, virtual drug screening,
docking prediction, molecular dynamics simulations, and ligand-binding affinities, as well
as the analysis of its pharmacokinetic properties.

2. Results
2.1. Literature Search
In this study, a bibliographic search was performed in the PubMed database for studies
that describe compounds isolated from Natural Peruvian products with antiviral properties.
The search resulted in 330 articles selected, whereas the time frame distribution among
the selected studies was a 70-year span (1950–2020). When conducting a co-occurrences
analysis of keywords, by setting the minimum number of keyword occurrences to five,
the number of keywords that meet the threshold was 134. While examining the network,
it is possible to notice that the keywords were organized into five clusters; the first cluster
(red color) keywords regarding the plant family “Asteraceae” and the genus “Lepidium”
were found associated with terms such as “antibacterial agents”, “anti-infective agents”,
and “antiprotozoal agents”. In the analysis, no “antiviral” activity related keyword was
found in any of the five clusters (Figure 1). Surprisingly, only two keywords related
to Peruvian plants were found in the analysis; this finding highlights a proportionally
small number of studies related to anti-microbial activity compared to the vast number of
described species of plants in Peru.

Figure 1. Bibliometric map created by VOSviewer based on MeSH terms co-occurrence.

Molecules 2021, 26, 3882 4 of 17

Antimicrobial agents with repurposing potential against viral diseases, such as te-
icoplanin, ivermectin, itraconazole, and nitazoxanide, have been described [35]. Consid-
ering this, compounds isolated from Smallanthus sonchifolius (commonly called Yacon),
a plant of the family Asteraceae, native to the Andean regions, that has been studied against
colon cancer, diabetes, and obesity [36], and Lepidium meyenii (commonly called Maca),
a root native to the Andean region, that has shown neuroprotective effects, antidepressant,
antioxidant, anticancer, and anti-inflammatory activities [37] were searched in the Pub-
Chem, Drug-Bank and ChEMBL databanks. In this regard, it was possible to find 14 and 26
compounds described from S. sonchilofolius (Table 1) and L. meyenii (Table 2), respectively.

Table 1. Natural compounds description of S. sonchilofolius.

PubChem ID Structure Name Part of the Plant/Extract TMW cLogP HBA HBB TPSA References

689043 Caffeic acid Roots/Methanolic extract 180.16 0.78 4 3 136.56 [38]

Chlorogenic
1794427 Roots/Methanolic extract 354.31 −0.768 9 6 245.29 [39]
acid

445858 Ferulic acid Roots 194.19 1.06 4 2 152.47 [40]

131753040 Sonchifolin Leaves/Methanolic extract 374.43 4.06 6 0 294.45 [41]

101324862 Polymatin B Leaves/NA 432.47 3.52 8 0 333.2 [42]

Leaves/dichloromethane
92043370 Uvedalin 448.47 2.09 9 0 335.55 [41]
extract
Molecules 2021, 26, 3882 5 of 17

Table 1. Cont.

PubChem ID Structure Name Part of the Plant/Extract TMW cLogP HBA HBB TPSA References

Leaves/dichloromethane
101250074 Fluctuanin 448.47 2.09 9 0 335.55 [43]
extract

Kaurenoic
73062 Leaves/NA 302.46 4.12 2 1 223.44 [44]
acid

370 Gallic acid Leaves/NA 170.12 0.11 5 4 116.41 [45]

5280805 Rutin Leaves/NA 610.52 −1.26 16 10 397.71 [45–48]

5281672 Myricetin Leaves/NA 318.24 1.14 8 6 208.29 [49]

5280863 Kaempferol Leaves/NA 286.24 1.84 6 4 195.59 [50]

5280343 Quercetin Leaves/NA 302.24 1.49 7 5 201.94 [51]

TMW: Total Molecular weight; iLOGP: octanol/water partition coefficient, HBAs: Number of H-Bond acceptors; HBDs: Number of H-bond
Donors; TPSA: molecular polar surface area.
Molecules 2021, 26, 3882 6 of 17

Table 2. Natural compounds description of L. meyenii.

PubChemID Structure Name Part of the Plant/Extract TMW cLogP HBA HBB TPSA References

Hypocotyl, roots/ethanol
656498 Glucotropaeolin 409.44 −0.75 10 5 270.57 [52,53]
extract

6602400 Glucosinalbin Hypocotyl/methanol extract 425.43 −1.09 11 6 276.92 [53,54]

5485207 Glucobrassicanapin Hypocotyl/methanol extract 387.43 −0.559 10 5 262.87 [53,55]

5317667 Glucobrassicin Hypocotyl/methanol extract 447.46 −1.957 11 5 295.92 [53,55]

N- Hypocotyl/hexane-ethanol
11198769 345.57 7.43 2 1 321.13 [56]
Benzylpalmitamide extract

N- Hypocotyl/hexane-ethanol
220495 373.62 8.34 2 1 348.65 [57]
Benzyloctadecanamide extract

N-Benzyl- Hypocotyl/hexane-ethanol
68742556 369.59 7.84 2 1 346.61 [58]
linoleamide extract

N-Benzyl-
Hypocotyl/hexane-ethanol
68741582 (9z,12z,15z)- 367.58 7.59 2 1 345.59 [59]
extract
octadecatrienamide
Molecules 2021, 26, 3882 7 of 17

Table 2. Cont.

PubChemID Structure Name Part of the Plant/Extract TMW cLogP HBA HBB TPSA References

5280450 Linoleic acid Hypocotyl/ether extracts 280.45 6.47 2 1 267.98 [48,60]

445639 Oleic acid Hypocotyl/ether extracts 282.47 6.72 2 1 269 [48,61]

71386083 7-Tridecanoic acid Hypocotyl/hexane extracts 212.33 4.45 2 1 200.2 [61]

TMW: Total Molecular weight; iLOGP: octanol/water partition coefficient, HBAs: Number of H-Bond acceptors; HBDs: Number of H-bond
Donors; TPSA: molecular polar surface area.

2.2. Virtual Screening

To find potential drug candidates among the 40 compounds related to S. sonchilofolius
and L. meyenii, a structure-based virtual screening approach against three major drug
targets of SARS-CoV-2, namely, Papain-like protease, N-terminal RNA binding domain
of nucleocapsid protein, and main protease, was performed. Results have shown that S.
sonchilofolius compounds presented statistically higher binding affinity when compared to
L. meyenii compounds in the three-drug targets (Figure 2A–C). Complementary, the calcu-
lation of the drug-likeness score using OSIRIS DataWarrior was also performed, and the
results have shown that only one compound, Rutin (PubChem ID: 5280805), presented a
positive score (Figure 2D–F).

Figure 2. Binding affinities of the molecules selected from S. sonchilofolius and L. meyenii toward
three main molecular targets of SARS-CoV2. Box plots with minimum and maximum values of
binding affinities for targets. Main protease (A), Papain-like protease (B), and N-terminal RNA
binding domain of nucleocapsid protein (C) are shown. Scatter plot showing binding affinities versus
drug-likeness score for main protease (D), Papain-like protease (E), and N-terminal RNA binding
domain of nucleocapsid protein (F) are shown.
Molecules 2021, 26, 3882 8 of 17

2.3. Molecular Dynamics Simulation Analysis

The potential drug prediction was conducted to the active site by docking prediction
in the PATCHDOCK server. The snapshot obtained from 50 ns of NPT simulation was used
for the calculation of the thermodynamics parameter.
The first analysis was the root mean squared deviation (RMSD) of the backbone;
this defined the similarity between two superimposed structures of backbone from PLpro,
Mpro N-terminal RNA binding domain of nucleocapsid protein. In Table 3, the average
RMSD values report a slight variation during the trajectory in the case of Mpro without
ligand (0.18 ± 0.04 nm). Indeed, the Mpro-rutin system has an average RMSD value
of 0.14 ± 0.01 nm, lower than Mpro without ligand. Herein, the ligand contributed to
the structural stability of Mpro begin 25 ns until the end of the simulation. In PLpro,
the average RMSD values indicated that the protein was less stable when it was bound to
the ligand. Therefore, it seems to achieve conformational changes in the PLpro structure.
However, the N-terminal RNA binding domain of nucleocapsid protein with Rutin shows
more structural stability.

Table 3. Average values of the parameters root mean squared deviation (RMSD), and radii of gyration
(Rg) analyzed for all trajectory (50 ns).

Mpro Mpro * Plpro PLpro * N N*

RMSD (nm) 0.18 ± 0.04 0.14 ± 0.01 0.52 ± 0.12 0.58 ± 0.14 0.17 ± 0.04 0.13 ± 0.02
RG (nm) 2.55 ± 0.01 2.55 ± 0.01 3.28 ± 0.05 3.24 ± 0.05 1.52 ± 0.01 1.53 ± 0.01
* Protein with Rutin.

The radii of gyration (Rg) analysis helped us verify the protein structures’ compaction,
where the average value for Mpro was similar in the two situations. As for the compaction
of PLpro, this is reduced if it is binding with the rutin molecule. On the other hand,
if we see the RG value of the N-rutin system, the compaction of this protein is increased.
In summary, these average RG values of the MD simulation showed that the rutin molecule
has a more significant effect on the PLpro structure than the other proteins.
Additionally, the average root mean squared fluctuations (RMSF) per residue of the
last 5 ns are represented in Figure 3. The Mpro-rutin system showed low fluctuations
concerning the system without ligand, for example, domain I of Mpro showed the most
significant reduction in fluctuations per residue (Mpro-rutin). This result can signal that
the Rutin contributes to the stability of the protein (Figure 3A). However, in the Plpro-rutin
system, high fluctuations per residue in the protein are observed; Figure 3B represents the
RMSF per residue of one of the three subunits of the Plpro complex docking. Figure 3C
shows the RMSF of the N protein without significant fluctuations.

2.4. MM/GBSA Estimation

We analyzed the MM/GBSA considering the last 500 snapshots from MD simulations
to estimate the binding free energy between the complexes studied. The results in Table 4
indicate the estimation of MM/GBSA in kcal/mol. In the Mpro-rutin system, there is a
more significant energy contribution by the van der Waals energies (−53,481 kcal/mol),
while Plpro-rutin and N-terminal RNA binding domain of nucleocapsid protein-rutin
systems were −21.713 kcal/mol and −34.342 kcal/mol, respectively. Furthermore, there is
a more significant contribution for the Plpro-rutin system (−47.024 kcal/mol) than the
other systems regarding electrostatic energy contribution. Furthermore, the contribution of
gas-phase energy to the total net energy of the system was significantly high in all systems
studies. Finally, the total binding energy in Mpro-rutin was better than in other systems
(−40.293 kcal/mol). However, in all systems is possible the action of Rutin.
Molecules 2021, 26, 3882 9 of 17

Figure 3. RMSF of C-alpha atoms per residue of protein with ligand (purple) and without ligand
(green). (A) RMSF for Mpro; (B) RMSF for PLpro; (C) RMSF for N protein.

Table 4. Binding free energies estimated for SARS-CoV-2 targets with Rutin by MM/GBSA method.

Mpro-Rutin Plpro-Rutin N-Rutin

Energy
Component Std Err of Std Err of Std Err of
Average Std Dev Average Std Dev Average Std Dev
Mean Mean Mean
VDWAALS −53.481 4.310 0.193 −21.713 6.213 0.278 −34.342 3.510 0.157
EEL −33.983 10.924 0.488 −47.024 23.706 1.059 −19.027 7.432 0.332
EGB 53.900 7.243 0.324 60.491 18.393 0.822 31.047 5.453 0.244
ESURF −6.730 0.402 0.018 −3.451 0.816 0.036 −4.684 0.371 0.017
∆ Ggas −87.463 11.289 0.504 −68.737 22.061 0.986 −53.370 8.381 0.374
∆ Gsolv 47.170 7.075 0.316 57.041 18.012 0.805 26.363 5.246 0.234
∆ TOTAL −40.293 5.740 0.256 −11.697 6.628 0.296 −27.007 4.115 0.184
VDWAALS = Van Der Waals energy; EEL = Electrostatic energy; EGB = Electrostatic contribution free energy calculated by Generalized
Born; ESURF = nonpolar contribution to the solvation free energy; ∆ Ggas = estimated binding free energy phase gas; ∆ Gsolv = estimates
binding free energy solvent; ∆ TOTAL = Estimated binding free energy. Values of energy in kcal/mol.
Molecules 2021, 26, 3882 10 of 17

Considering the results of the molecular dynamics simulations and the energy estima-
tion by the MM/GBSA method, we found that the Mpro-rutin system was more stable than
PLpro and N-terminal RNA binding domain of nucleocapsid protein. Thus, we tested an
HIV protease inhibitor (Lopinavir) in order to compare it with the best candidate obtained
by virtual screening of this work.
The molecular structure of Lopinavir was downloaded from Protein Data Bank by the
access code PDB ID: AB1. The ACPYPE server generated the topologies of Lopinavir in the
AMBER force field like the methodology for Rutin.
In principle, molecular dynamics simulation was applied to analyze the behavior
of the Mpro-lopinavir system. The same computational details proposed in the method-
ology for molecular dynamics simulation of Mpro-rutin were used. Figure 4 shows the
RMSD analysis, which indicates that both ligands show similar effects on the structural
stabilization of Mpro. In addition, we were able to analyze the binding free energy of Mpro-
lopinavir through MM/GBSA. Our results indicated that rutin showed better binding free
energy (−40.293 ± 5.740 kcal/mol) than Lopinavir (−32.7810 ± 1.8461 kcal/mol).

Figure 4. Comparison of molecular dynamics simulation trajectory between Rutin and Lopinavir.
(A) SARS-CoV-2 Mpro with Lopinavir in the last frame of the MD simulations. (B) SARS-CoV-2 with
Rutin in the last frame of the MD simulations. (C) RMSD plotting of backbone of SARS-CoV-2 Mpro.

3. Discussion
Peru is one of the privileged countries with incredible biodiversity. However, there are
relatively few studies on the antimicrobial and antiviral effect of metabolites from these
sources. Based on the richness of Peruvian plants, the present study aimed to perform
virtual screening of various isolated natural compounds against SARS-Cov-2 therapeutic
targets. Our results showed the promising effect of a compound found on S. sonchilofolius,
Molecules 2021, 26, 3882 11 of 17

called Rutin, against three SARS-CoV-2 targets: Main Protease, Papain Like Protease,
and N-terminal RNA binding domain of the nucleocapsid protein. Rutin is a flavonol
widely present in various fruits and vegetables [62], with antioxidant, anti-inflammatory,
and antiviral effects [63].
Previous reports have shown the potential use of plants and isolated compounds
against SARS-CoV targets and other human coronaviruses. Terpenoids and lignoids iso-
lated from Chamaecyparis obtuse var. formosana Hayata, Juniperus formosana Hayata, and Cryp-
tomeria japonica (Thunb. ex L.f.) D.Do showed the ability to inhibit viral replication [64];
the flavonoids rhoifolin, herbacetin, and pectolinarin, from a flavonoid library, showed
noteworthy inhibitory activity against SARS-CoV virus [65]; and epigallocatechin gallate
and gallocatechin gallate also inhibited SARS-CoV 3CLpro. However, selecting compounds
for further clinical assessment should be carefully considered, and it is crucial that in vitro
and in silico experimental evidence be conclusive [66].
Smallanthus sonchifolius, also known as yacón, is a perennial hemicryptophyte tuberous
shrub native to the South American Andes, widely cultivated for its nutraceutical and
medicinal properties [67]. Several studies had shown that the consumption of yacón has
improved parameters related to type II diabetes, improved insulin production and de-
creased blood glucose levels. Furthermore, the anti-inflammatory and antioxidant activities
of organic and aqueous extracts of yacón tubers and aerial parts have been described [68].
Furthermore, note the antibacterial potential of the constituents of S. sonchifolius against
methicillin-resistant Staphylococcus aureus [69], Bacillus subtilis, and Pyricularia oryzae [41].
In recent years, computational simulations allowed the improvement of structural
modelling of macromolecules. This study compared the molecular dynamics of Rutin
against three SARS-CoV-2 therapeutic targets (Mpro, Plpro and N-terminal RNA binding
domain of nucleocapsid protein). Mpro is a homodimer enzyme with a catalytic dyad
composed of Cys145 and His41 [70]. Our results show that Rutin has remained interacting
in the catalytic dyad of Mpro with Asn142, Met49, His41, and Asp187. Our results agree
with Jin and collaborators’ findings, where they found that the N3 inhibitor interacts with
the residues Asn142, Met49, His41, Cys145, Gln189, Val3, Leu4, Leu27, and Thr25 [71].
In this computational approach, the Rutin showed a high probability of binding to Mpro.
To date, there is no drug specifically formulated for the treatment of Covid-19 and
approved by the FDA. Therefore, specific candidates were repurposed from their actual
use among the best results, such as the antiretrovirals boceprevir, asunaprevir, narlaprevir,
paritaprevir, and telaprevir, used for the hepatitis C and Lopinavir for HIV treatments,
whose effect on the protein Mpro are still being evaluated [72,73]. In our study, when we
compared the inhibitory effect of Lopinavir to Rutin, we observed a similar positive effect
between both compounds within the Mpro pocket, confirming the potential of Rutin against
SARS-CoV-2 Mpro.
In parallel, it is reported that among candidates in clinical trials, such as protease in-
hibitor GC376 and ketone-based inhibitors showed promising action due to their interaction
with dyad catalytic (His 41 and Cys145) [74–76].
On the other hand, Plpro is a therapeutic target composed of three protein subunits.
Previous studies of the crystal structure of Plpro bound to an inhibitor showed that the
binding site was composed of Tyr268, Asp164, and Gln269 [77]. The molecular dynamics
simulation of the Plpro-rutin complex demonstrated that Rutin maintains an interaction
with the Tyr268 and Gln269 residues comparable with experimental methods. Regarding
the N-terminal RNA binding domain of nucleocapsid protein, we note that the Rutin
maintains a coupling that allows the fluctuations reduction in this protein domain. In all
cases, the estimation of the MM/GBSA showed favorable binding free energy in the
complexes analyzed, where the best energy is observed for the Mpro-rutin. In addition,
this system was the most stable system during the molecular dynamics simulations.
Molecules 2021, 26, 3882 12 of 17

4. Materials and Methods

4.1. Literature Search Strategy and Data Collection
The bibliographic extraction was performed from the National Center for Biotechnol-
ogy Information (NCBI) databases. PubMed (https://www.ncbi.nlm.nih.gov/pubmed/,
accessed on 23 December 2020) is a free resource database and provides uniform indexing
of biomedical literature, the Medical Subject Headings (MeSH terms), which form a con-
trolled vocabulary or specific set of terms that describe the topic of a paper consistently
and uniformly [78]. In this regard, MeSH terms were employed in the string query to
improve the search accuracy. The data set was retrieved from PubMed on 23 December
2020, based on the following search string: “Peru” [MeSH Terms] AND “Natural prod-
ucts” [MeSH Terms]. To search among the results for Peruvian natural products with
antiviral proprieties, the co-occurrence network map of MeSH terms was created using the
VOSviewer server (Version 1.6.16) [79].
The simplified molecular-input line-entry system (SMILE) of compounds previously
described in the natural products selected in the previous step was searched and retrieved
from PubChem [80], DrugBank [81], or ChEMBL [82] servers. The following physicochemi-
cal properties, Total Molecular weight (MW), octanol/water partition coefficient (iLOGP),
Number of H-Bond acceptors (HBAs), Number of H-bond Donors (HBDs), and the molec-
ular polar surface area (TPSA), for each compound were calculated within the Osiris
DataWarrior v05.02.01 software [83].

4.2. Virtual Screening

The FASTA sequences of the drug targets Papain-like protease (PLpro) (PDB ID:
6W9C), N-terminal RNA binding domain of nucleocapsid protein (N) (PDB ID: 6M3M) and
main protease (Mpro) (PDB ID: 6LU7) of SARS-CoV-2 were retrieved from Protein Data
Bank (https://www.rcsb.org, accessed on 23 December 2020), and subject to automated
modeling in SWISS-MODEL [84]. Furthermore, the compounds were imported into Open-
Babel within the Python Prescription Virtual Screening Tool (PyRx) [85] and subjected to
energy minimization. PyRx performs structure-based virtual screening applying docking
simulations using the AutoDock Vina tool [86], whereas the drug targets were uploaded as
macromolecules. For the analysis, the search space encompassed the whole of the modeled
3D models; and the docking simulation was then run at exhaustiveness of 8 and set to only
output the lowest energy pose.
The Osiris DataWarrior software was employed to calculate the drug-likeness score
of each compound; the calculation is based on a library of ~5300 substructure fragments
and their associated drug-likeness scores. This library was prepared by fragmenting
3300 commercial drugs as well as 15,000 commercial non-drug-like Fluka compounds.
Furthermore, the potential tumorigenic, mutagenic, and irritant action of each compound
was predicted by comparison to a precompiled fragment library derived from the RTECS
(Registry of Toxic Effects of Chemical Substances) database [83].

4.3. Molecular Dynamics Simulations and Molecular Mechanics Generalized Born Surface
Area Calculations
The protein structures downloaded were reviewed in a Text Editor, and the molecules
that are not necessary for calculations were deleted. Likewise, based on homology modeling
in the Swiss-Model server (https://swissmodel.expasy.org, accessed on 12 January 2021),
it was possible to add the missing residues to complete the protein structures. In the case
of PLpro, the Zn ions’ coordination with the SG atom of Cys270, Cys224, Cys189, Cys192,
and Cys226 was considered.
The automatic generation topologies with the AMBER force field for the rutin molecule
were obtained from ACPYPE server [87]. It was selected the charge method semi-empirical
AM1-BCC, parameterized to reproduce HF/6-31G * RESP charges. Consequently, molecu-
lar dynamics (MD) simulations were carried out in GROMACS v. 2020 [88]. We considered
AMBER99SB-ILDN force field, TIP4P water model, and ions to neutralize the system.
Molecules 2021, 26, 3882 13 of 17

The conditions of simulations consisted of three steps: the first was the minimization with
the steepest descent algorithm for 50,000 steps, the second was the MD of the equilibrium
in the NVT canonical ensemble with V-rescale thermostat at 309.65 K with a time of calcu-
lation of 10 ns, and the third step was the MD production without restraint condition in
the NPT isobaric-isothermal ensemble with V-rescale thermostat at 309.65 K and Parrinello–
Rahman barostat at 1 bar of reference pressure by 50 ns. The thermodynamic parameters
were analyzed using the Gromacs tools and the plotting graph realized with Gnuplot v5
package [89].
To determine the binding free-energy of receptor–ligand, we used the Molecular
Mechanics Generalized Born Surface Area (MM/GBSA) using gmx MMPBSA v1.4.1 [90]
tool based on MMPBSA. py [91] From AmberTools20 suite. The molecular visualization of
the interaction of the complex receptor–ligand structure was carried out using the VMD
v1.9.4 (Visual Molecular Dynamics) software [92], and the 2D diagrams of receptor–ligand
were visualized using the PoseView server [93].

5. Conclusions
This work has analyzed many data reported in PubMed data based on natural com-
pounds from Peruvian plants with antiviral activity. After screening, our results showed
that Smallanthus sonchilofolius and Lepidium meyenii were the most outstanding options
due to their antibacterial, anti-infective, anti-inflammatory, antineoplastic, antioxidant,
and antiprotozoal effect. Considering these native plants, we found a total of 40 reported
compounds, where the Rutin compound showed high binding affinity against three thera-
peutic targets of SARS-Cov-2: Main Protease, Papain Like Protease, and N-terminal RNA
binding domain of the nucleocapsid protein. To complement this work, computational
simulation methods with explicit solvent were employed. Our results demonstrated that
the Mpro-rutin system had the best coupling during the molecular dynamics simulation.
Moreover, the MM/GBSA free energy calculations showed that the Mpro-rutin complex
had the highest energy. Therefore, we propose the potential use of Peruvian native plants,
like Smallanthus sonchilofolius, for further in vitro and in vivo studies in search of new
alternatives in the treatment of COVID-19 that could be used in future drug formulations.

Author Contributions: Conceptualization, L.D.G.-M. and H.L.B.-C. and M.A.C.-F.; methodology,

L.D.G.-M., H.L.B.-C., K.M.-U. and M.A.C.-F.; validation, K.M.-U.; investigation, L.D.G.-M. and H.L.B.-
C.; writing—original draft preparation, L.D.G.-M., H.L.B.-C. and M.A.C.-F.; writing—review and
editing, L.D.G.-M., H.L.B.-C. and M.A.C.-F. All authors have read and agreed to the published version
of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: We appreciate the computational support of the internal project UCSM 7309-CU-
2020. We would like to express our admiration and acknowledgments to the health care personnel
worldwide and our sincerest condolences to those who suffered from the COVID-19 pandemic.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are not available from the authors.

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